The invention relates to laser scanning cytometry. More particularly, the invention relates to a system for automatically focusing a laser scanning cytometer on a substrate.
Laser scanning cytometers can be used for detecting and measuring various properties of a biological sample. Typically, the sample is located on a substrate and stained with a fluorescent dye which binds to a particular cell constituent. An objective lens emits a laser beam onto the sample. Each fluorescently marked cell illuminated by the laser beam emits a pulse of fluorescent light. The intensity of the pulse of fluorescent light depends on the amount of fluorescent dye contained in the cell. Typically, a detector measures the fluorescent intensity of each cell. From the intensity of the fluorescent light pulse, a processor can determine the amount of the particular cell constituent present in the sample. Some laser scanning cytometers employ a plurality of dyes, each selected to bind to a different cell constituent. Laser scanning cytometers may also illuminate the sample at a plurality of wavelengths to determine the presence and/or concentration of multiple cellular constituents and other features of the cells contained in the sample.
Typically, when a laser scanning cytometer scans a sample on a surface of a substrate, the objective lens has a relatively narrow depth of focus (i.e., a focus window). If the substrate surface falls outside of the depth of focus window, cell constituent detection is less than optimal. A variety of factors can cause at least portions of the substrate surface to fall outside of the depth of focus window. For example, manufacturing imperfections in the substrate upon which the sample is located, and tolerance imperfections in mechanisms that hold the substrate in place, can cause the surface of the substrate to be tilted or displaced relative to the focal plane of the objective lens.
Accordingly, one object of the invention is to provide a system for determining and maintaining a more optimal relationship between the relative positions of the objective lens and the substrate holding the sample to be analyzed.
The invention relates to a method and system for focusing a laser scanning cytometer. A single laser is used for taking cytometric measurements and for focusing the cytometer. Laser light is transmitted though an objective lens and directed at a substrate to cause markers in a sample to fluoresce. A byproduct of cytometric measurements is that a portion of the laser light incident on the substrate is backscattered and collected by the objective along with the fluorescing light. This backscattered laser light is separated from the fluorescent signal and used to focus the objective.
According to one embodiment, the invention relates to a method of determining an optimal focal distance. The method includes transmitting laser light through an objective lens at a plurality of focal distances onto a substrate at a plurality of locations. The objective lens receives a plurality of reflected components of the laser light at each of the plurality of focal distances at each of the plurality of locations. The plurality of reflected components received at each of the plurality of focal distances at each of the plurality of locations are processed to determine which focal distance is the optimal focal distance for each of the plurality of locations. The laser light and the objective lens also enable focus measurements to be made with or without a test sample placed on the substrate.
In one aspect, the method includes processing the optimal focal distance for at least a portion of the plurality of locations to determine a focal plane, wherein the focal plane provides a planar approximation of the optimal focal distance for locations on the substrate in addition to the plurality of locations.
In another aspect, the method includes defining a plurality of scan fields on the substrate, wherein each of the scan fields contains at least one of the plurality of locations. The optimal focal distance is selected in each of the plurality of scan fields to be a representative focal distance for each of the plurality of scan fields. The representative focal distance for each of the plurality of scan fields is processed to generate a map of the representative focal distance for each of the plurality of scan fields. At least one of the plurality of locations within each of the plurality of scan fields is the geometric center of the scan field.
In another aspect, the method includes interpolating between the optimal focal distance for ones of the plurality of locations to generate a map representative of an approximation of the optimal focal distance for locations on the substrate in addition to the plurality of locations.
In still another aspect, the method includes scanning the laser beam through the objective lens at each of the plurality of focal distances onto the substrate and through each of the plurality of locations to generate a scan line corresponding to each of the plurality of focal distances at each of the plurality of locations. The objective lens then receives the plurality of reflected components, wherein each of the plurality of reflected components is received at each of the corresponding focal distances and from a plurality of scan locations along a scan line corresponding to one of the plurality of locations. The plurality of reflected components associated with each of the scan locations along each of the scan lines at each of the plurality of focal distances are then processed to determine the optimal focal distance for each of the plurality of locations. The reflected components from a plurality of the scan lines corresponding to one of the plurality of focal distances for each of the plurality of the focal distances can be averaged to determine the optimal focal distance for each of the plurality of locations. The reflected components of each one of the plurality of scan lines can be processed to obtain the characteristics of a spatial frequency spectrum for each one of the plurality of scan lines to determine the optimal focal distance for each of the plurality of locations. Such characteristics include power at a specific frequency or the power sum over a band of frequencies. The reflected components of each one of the plurality of scan lines can also be processed to obtain a normalized value for each one of the plurality of scan lines to determine the optimal focal distance for each of the plurality of locations. According to one aspect, the laser light is moved along the scan line with a galvanometer-controlled mirror and the plurality of reflected components are collected with a photo-detector.
According to another embodiment, the invention relates to a system for determining an optimal focal distance. The system includes a laser source adapted to provide laser light, an objective, and a processor. The objective is adapted to direct said laser light at a plurality of focal distances onto a substrate at a plurality of locations, and to receive a plurality of reflected components of the laser light at each of the plurality of locations. The objective receives reflected components at a corresponding one of the plurality of focal distances, and from a corresponding one of the plurality of locations. The processor is adapted to process the plurality of reflected components corresponding to each of the plurality of focal distances at each of the plurality of locations to determine the optimal focal distance for each of the plurality of locations.
According to one feature, the processor is adapted to select one of the plurality of focal distances as the optimal focal distance and to process the optimal focal distance for at least a portion of the plurality of locations to determine a focal plane, wherein the focal plane provides a planar approximation of the optimal focal distance for locations on the substrate in addition to the plurality of locations.
According to another feature, the processor is adapted to define a plurality of scan fields on the substrate, wherein each of the scan fields contains at least one of the plurality of locations. The processor selects the optimal focal distance for the at least one of the plurality of locations in each of the plurality of scan fields to be a representative focal distance for each of the plurality of scan fields. The processor processes the representative focal distance for each of the plurality of scan fields to generate a map of the representative focal distance for each of the plurality of scan fields. Additionally, the processor selects the at least one of the plurality of locations to be at a geometric center of each of the plurality of scan fields.
According to another feature, the processor is adapted to interpolate between the optimal focal distance for ones of the plurality of locations to generate a map representative of an approximation of the optimal focal distance for locations on the substrate in addition to the plurality of locations.
According to still another feature, the laser source is adapted to scan the laser beam through the objective at each of the plurality of focal distances onto the substrate and through each of the plurality of locations to generate a scan line corresponding with each of the plurality focal distances at each of the plurality of locations. Also, the objective is adapted to receive the plurality of reflected components, wherein each of the plurality of reflected components is received at the corresponding one of the plurality of focal distances and from a plurality of scan locations along a scan line corresponding to one of the plurality of locations. Additionally, the processor is adapted to process the plurality of reflected components associated with each of the scan locations along each of the scan lines at each of the plurality of focal distances to determine the optimal focal distance for each of the plurality of locations.
According to another feature, the processor is also adapted to average the reflected components from a plurality of the scan lines corresponding to one of the plurality of focal distances for each of the plurality of the focal distances to determine the optimal focal distance for each of the plurality of locations. According to a further feature, the processor is adapted to process the reflected components of each one of the plurality of scan lines to obtain the characteristics of a spatial frequency spectrum for each one of the plurality of scan lines to determine the optimal focal distance for each of the plurality of locations. According to a further feature, the processor is adapted to process the reflected components of each one of the plurality of scan lines to obtain a normalized value for each one of the plurality of scan lines to determine the optimal focal distance for each of the plurality of locations.
According to another embodiment, the invention relates to a method for performing a laser-based measurement. The method includes transmitting laser light through an objective along an optical axis at a plurality of focal distances onto a substrate at a plurality of locations. The objective receives a plurality of reflected components of the laser light at each of the plurality of locations, wherein each of the plurality of reflected components is received at a corresponding one of the plurality of focal distances and from a corresponding one of the plurality of locations. The plurality of reflected components corresponding to each of the plurality of focal distances at each of the plurality of locations are processed to determine the optimal focal distance for each of the plurality of locations. A representation of the optimal focal distance for each of the plurality of locations is stored. According to another feature, the method includes introducing a sample onto the substrate and measuring a characteristic of the sample by selecting a measurement location on the substrate, selecting a focal distance for the objective based at least in part on the stored representation, and performing a laser-based measurement on the sample at the measurement location and at the selected focal distance. The measuring step can be performed at additional measurement locations.
According to one aspect, the method includes performing the laser measurement at least in part by measuring a fluorescence characteristic of the sample and selecting one of the plurality of focal distances as the optimal focal distance. According to another aspect, the method includes processing the optimal focal distance for at least a portion of the plurality of locations to determine a focal plane, wherein the focal plane provides a planar approximation of the optimal focal distance for locations on the substrate in addition to the plurality of locations. According to another feature, the method includes defining a plurality of scan fields on the substrate, wherein each of the scan fields contains at least one of the plurality of locations. According to another feature, the method includes selecting the optimal focal distance for the at least one of the plurality of locations in each of the plurality of scan fields to be a representative focal distance for each of the plurality of scan fields. The representative focal distance for each of the plurality of scan fields is processed to generate a map of the representative focal distance for each of the plurality of scan fields. At least one of the plurality of locations within each of the plurality of scan fields is at a geometric center of each scan field.
According to another feature, the method includes interpolating between the optimal focal distance for ones of the plurality of locations to generate a map representative of an approximation of the optimal focal distance for locations on the substrate in addition to the plurality of locations.
According to another feature, the method includes scanning the laser beam through the objective at each of the plurality of focal distances onto the substrate and through each of the plurality of locations to generate a scan line corresponding with each of the plurality of focal distances at each of the plurality of locations. According to a further feature, the method includes receiving the plurality of reflected components, wherein each of the plurality of reflected components is received at the corresponding one of the plurality of focal distances and from a plurality of scan locations along a scan line corresponding to one of the plurality of locations. The plurality of reflected components associated with each of the scan locations along each of the scan lines at each of the plurality of focal distances are processed to determine the optimal focal distance for each of the plurality of locations. The reflected components from a plurality of the scan lines corresponding to one of the plurality of focal distances for each of the plurality of the focal distances can be averaged to determine the optimal focal distance for each of the plurality of locations. The reflected components of each one of the plurality of scan lines can be processed to obtain a spatial frequency for each one of the plurality of scan lines to determine the optimal focal distance for each of the plurality of locations. The reflected components of each one of the plurality of scan lines can be processed to obtain a normalized value for each one of the plurality of scan lines to determine the optimal focal distance for each of the plurality of locations. The laser light is moved along the scan line with a galvanometer-controlled mirror and the plurality of reflected components is collected with a photo-detector.